Assessment of the diet of the critically endangered northern hairy‐nosed wombat (Lasiorhinus krefftii) using DNA metabarcoding

Abstract Invasive buffel grass (Cenchrus ciliaris) is considered a threat to the critically endangered northern hairy‐nosed wombat (Lasiorhinus krefftii; NHW). Buffel grass outcompetes native grasses, reducing availability of native food items for NHW, and causes more intense fires due to the large volumes of dead matter it produces. Previous studies suggested buffel grass was increasing in the diet; however, the diet of the NHW has not been reassessed for over two decades and was limited to Epping Forest National Park, with the population at Richard Underwood Nature Refuge having never been assessed. The recently released 2022 Recovery Action Plan for the species outlined objectives to assist its conservation and recommended the impact of buffel grass on the species' diet be investigated. This study aimed to determine: (1) which plant species are being consumed by the NHW; (2) the differences in the diet between sites; (3) differences between seasons; and (4) the abundance of buffel grass in the diet. The diet was assessed using DNA metabarcoding of scat samples collected from both sites from winter 2020 to spring 2021. Site and season significantly affected the diet of the NHW. Buffel grass dominated the diet and has increased in the diet since past assessments. The findings of this study will support population and habitat management of the critically endangered NHW. Enhanced knowledge of dietary items consumed at both sites will also assist efforts to locate additional sites suitable for translocation.


| INTRODUC TI ON
The diet of a herbivore is determined by a combination of physiological and environmental factors, including food availability, gut morphology and nutritional requirements (Gordon & Illius, 1996;Illius & Gordon, 1992;Kohlmann & Rosenhoover, 1994).The diet of many herbivores, particularly large herbivores, is dominated by grasses, sedges, forbs and woody dicots (du Toit & Olff, 2014).Herbivores may consume a combination of dietary items that offer differing quantities of nutrients to fulfil specific requirements and achieve nutrient balance (Dussutour et al., 2010;Felton et al., 2009;Raubenheimer et al., 2005;Robbins et al., 2007;Rothman et al., 2011;Simpson et al., 2004).Although some factors that influence diet such as gut morphology cannot be manipulated, others such as food availability can, allowing conservationists to implement strategies to influence the diet of vulnerable or threatened species to improve their nutritional health.For herbivores, conservation strategies may involve valuable plant food items being promoted within their habitat while nondietary items may be controlled (Lindsay & Cunningham, 2011).
Lasiorhinus krefftii, known as the northern hairy-nosed wombat (NHW), is a critically endangered (Taggart et al., 2016) herbivorous marsupial native to Australia.There are ~315 individuals living across two populations: ~300 at Epping Forest National Park (EFNP) and ~15 at Richard Underwood Nature Refuge (RUNR), a translocated population established in 2009 (Department of Environment andScience, 2021, 2022).The diet of the NHW at EFNP has been assessed in the past using histological techniques (Crossman, 1988;Flosser, 1986;Woolnough, 1998), but has not yet been assessed using DNA metabarcoding techniques, like the closely related southern hairy-nosed wombat (Lasiorhinus latifrons; Sobek & Walker, 2021).Woolnough (1998) assessed the diet of the NHW using stable isotopes and long-chain alkanes, but to minimise bias introduced by varying analyses, comparisons made throughout this study will refer to the results of histological analysis only.In this study, grass species constituted a large proportion of the diet, particularly Heteropogon, Enneapogon and Aristida species.
Introduced Cenchrus ciliaris, otherwise known as buffel grass, was also identified as a dietary item.A variety of forbs complemented the diet, as well as the sedge Fimbristylis dichotoma (Woolnough, 1998).The diet of the NHW population at RUNR has never been assessed.
DNA metabarcoding is more specific and faster than traditional histological studies, allowing a greater number of samples to be processed in a shorter amount of time.It also allows for a lower resolution of taxonomic level to be identified, which is more difficult to determine using microscopic identification methods as some species are morphologically very similar at the species or Genus level, and can undergo changes due to digestion (Kaunisto et al., 2017;Valentini et al., 2009).
DNA metabarcoding also is more likely to identify cryptic species, as these species are less likely to be chosen as reference material in histological studies (Thongtam na Ayudhaya et al., 2017).

Comparisons of previous NHW diet studies indicate that the diet
has changed over time (Flosser, 1986;Woolnough, 1998) with some changes being linked to changes in forage availability and floristic composition due to severe droughts (Horsup, 1998).Other dietary changes, however, have been linked to the increase in abundance of buffel grass throughout EFNP (Horsup, 2004), where it is now the dominant grass species, occurring more frequently and contributing more to biomass than any other dietary item (Woolnough, 1998).
The increase in buffel grass at EFNP was reported to have been followed by a dramatic increase in buffel grass in the diet of the NHW (Horsup, 2004).The abundance of buffel grass increased from ~1% in 1982-1983 (Flosser, 1986) to up to 27% during 1993-1996(Woolnough, 1998)).The occurrence of buffel grass within the floristic community at RUNR has also reportedly increased (Department of Environment and Science, 2022).This history of changes in the diet of the NHW suggests the diet may have again changed substantially since the most recent assessment over two decades ago.
The NHW Recovery Action Plan 2022 (Department of Environment and Science, 2022) outlined that one of four major objectives of the recovery plan is to fill gaps in our knowledge of the NHW; one knowledge gap identified as requiring investigation was the impact of buffel grass on the diet of the NHW.Buffel grass is considered a threat to the NHW as it has changed the composition of the vegetation community at both EFNP and RUNR, in turn changing the populations' food resources (Department of Environment and Science, 2022).Buffel grass grows and invades new habitats quickly and significantly outcompetes native Australian grasses, reducing overall species richness within an area (Marshall et al., 2012;Miller et al., 2010).Buffel grass causes hotter and faster fires because it produces large volumes of standing dead matter, more so than Australian native grasses.Soil disturbance caused by burrow building by NHW allows buffel grass to easily establish around burrows and outcompete native grasses, which in turn causes wombats to forage further from their burrow (and hence refuge) to find food (Marshall et al., 2012).An updated assessment of the current diet of the NHW will inform management decisions, including strategies such as controlling potentially threatening forage taxa, for example buffel grass or promoting native endemic food items, as previously described in Woolnough (1998).Improved access to food items of sufficient nutritional quality could support successful reproduction (Trites & Donnelly, 2003) and survival rates (Hutchings et al., 2003;Lochmiller & Deerenberg, 2000) of the NHW, thus supporting population growth.
This project aimed to determine the: (1) plant species being consumed by the NHW using modern DNA metabarcoding techniques, (2) differences in the diet across the two sites where NHW live, (3) seasonal changes to diet choice and (4) abundance of buffel grass in the diet.

| Study area
The two NHW population sites, EFNP and RUNR, were the focus areas for this study.EFNP is a 2750 hectare area of open eucalypt woodland within the Brigalow belt (22°21′ S and 146°41′ E), central Queensland (Department of Environment and Science, 2022).RUNR is a 130 hectare area of eucalypt woodland along river levees at Yarran Downs, southern Queensland (Department of Environment and Science, 2022).A1).There are approximately 300 NHW at EFNP and 15 at RUNR with 20 burrows recognised as 'primary' burrows frequently occupied by wombats at RUNR (Department of Environment and Science, 2022;Jørgensen et al., 2021).Except for four historic scat samples collected from EFNP in 2013EFNP in , 2014EFNP in , 2015EFNP in and 2017, all , all other scats were collected from near burrow entrances and all pellets were collected (as opposed to a single pellet from a group).One pellet was kept for assessment in this study, and the remainder were used for other research (Casey et al., unpub.).Scats were selected based on freshness as indicated by moistness (Banks et al., 2002), and effort was made to avoid contamination from substrates such as dirt (McInnes et al., 2017) and insects (dung beetles).The date, time and GPS coordinates for all scats collected, and the closet burrow number, were recorded.Scats were stored in paper bags at −80°C until analysis, on average scats were in the paper bag 2 h before being frozen.(Ravi et al., 2018) and sequence data generated using the Illumina bcl2fastq 2.20 pipeline.

| DNA extraction, sequencing and bioinformatics
Sterile water was run as a negative control.
The bioinformatics analysis consisted of demultiplexing, quality control, Amplicon Sequence Variant (ASV) calling and taxonomic classification.Diversity profiling analysis was performed with QIIME 22019.7 (Bolyen et al., 2019).Demultiplexed raw reads were primer trimmed and quality filtered using the cutadapt plugin, and denoising was performed with DADA2 (Callahan et al., 2016;via q2-dada2).

| Statistical analyses
The data were summarised as relative read abundance (RRA) and frequency of occurrence (FOO).RRA referred to the percentage of total reads a taxon contributes per sample and was used as an indication of the abundance of taxa in the diet in this study (Deagle et al., 2019).FOO referred to the percentage of samples in which a taxon was detected.A PERMANOVA was performed to detect whether the abundance of each taxon in the diet was affected by site and season.A zero-adjusted Bray-Curtis dissimilarity index was calculated for each site, and an ANOSIM (analysis of similarities) was used to detect whether the diet varied between seasons at each site, with 9999 permutations of the test statistic.SIMPER (Similarity percentages) was used to determine the average dissimilarity between seasons at each site and how much of a contribution (%) each taxon made to the dissimilarity.Analyses were conducted using Primer 7 (Clark & Warwick, 2001).

| RE SULTS
Of the total 85 scats available for sequencing, we were unable to At EFNP 20 orders, 32 families, 80 genera and 87 species, and at RUNR 20 orders, 33 families, 87 genera and 72 species of plant were identified in the scats.
At EFNP, eight taxa returned an abundance of ≥0.5%, including unidentified Tribulus spp.(Figure 3) which was not present in the diet at RUNR.Twelve taxa returned an abundance of ≥0.5% at RUNR, including Eragrostis curvula and unidentified Chloris spp.which were not present in the diet at EFNP.Four taxa contributed ≥0.5% at both sites: buffel grass, Boerhavia erecta, Evolvulus alsinoides and Brachiaria subquadripara.Buffel grass was the most abundant taxon in the diet at both sites; the average abundance of buffel grass in the diet at EFNP was 71.6%, and 68.6% at RUNR (Figure 3).
The PERMANOVA revealed that there was a significant interaction between site and season affecting the abundance of items within the diet (p ≤ .05).The pairwise SIMPER revealed the two greatest contributors to dissimilarity between the two sites were the mean abundance of buffel grass, which was greater at EFNP compared with RUNR, followed by the mean abundance of unidentified Poaceae spp., which was greater at RUNR compared with EFNP (Fig- ure 3).The next six greatest contributors to dissimilarity between the two sites were Chrysopogon latifolius, Boerhavia erecta, Brachiaria subquadripara, Perotis hordeiformis, unidentified species of the Fimbristylis genus and unidentified species of the Tribulus genus, which were all more abundant in the diet at EFNP compared with RUNR (Figure 3).

| Seasonal differences at EFNP
At EFNP, buffel grass returned the highest abundance of all taxa during every season; the average abundance of buffel grass was greater during winter 2020 (92.6%) and winter 2021 (92.5%) compared with spring 2020 (76.0%) and summer 2020/2021 (30.8%; Figure 4).A2).Buffel grass and unassigned spp.were the only two taxa with a FOO of 100% during winter 2021, while the FOO of unidentified Malvaceae spp.and Evolvulus alsinoides was 86% and 63%, respectively (Appendix Table A2).
Within those three significantly different pairwise comparisons, buffel grass, Chrysopogon latifolius and Boerhavia erecta were the three highest contributors to dissimilarity (Table 1).
The one-way ANOSIM pairwise tests revealed that there was no significant difference in the diet between 2013, 2014, 2017 and any other sampling period at EFNP (p = .07-.50).However, the mean F I G U R E 4 Proportional abundance of taxa in the diet of northern hairy-nosed wombat at Epping Forest National Park, in total and across each season.All taxa that did not contribute ≥0.5% within at least one season are combined and represented as 'Other'.

TA B L E 1
Pairwise SIMPER results including the 10 taxa with the greatest contribution to dissimilarity for significant pairwise ANOSIM tests from Epping Forest National Park (EFNP).
At RUNR, the one-way ANOSIM among seasons revealed a significant effect of season on the diet (R = 0.305, p ≤ .05),with the pairwise tests revealing significant differences between all seasons (R = 0.232-0.560,p ≤ .05),except between summer 2020/2021 and winter 2020, and summer 2020/2021 and winter 2021.The pairwise SIMPER revealed the pairwise comparisons with the four highest average dissimilarities all involved autumn 2021, and within all significant pairwise tests at RUNR, buffel grass and Poaceae spp.were the two greatest contributors to dissimilarity (Table 3).

| DISCUSS ION
The diet of the NHW varied significantly seasonally and between EFNP and RUNR.Buffel grass dominated the diet of the NHW at both sites.Only eight taxa at EFNP and 12 taxa at RUNR contributed more than ≥0.5% each to the diet (Figure 3).There was a high number of taxa detected within the diet (191 taxa) but few primary dietary items, with the vast majority contributing <0.5% to dietary abundance (93% of taxa) and having a FOO <20% (84% of taxa).The taxa that did contribute ≥0.5% to dietary abundance often also had a higher FOO than most other taxa.Buffel grass heavily dominated the diet of the NHW, with a FOO of 100% and an abundance almost 10 times greater than the next most abundant taxon.
Despite buffel grass dominating the diet at both sites, there was significant difference in the diet of the NHW between EFNP and RUNR.Approximately two thirds of the taxa identified were present in the diet at one site but not the other, including some primary dietary items (contributed ≥0.5% to diet abundance within a site), such as Tribulus spp., Eragrostis curvula and Chloris spp.In addition, numerous taxa were primary dietary items at one site, but were of low abundance in the diet at the other, including Chrysopogon latifolius and unidentified Poaceae spp.(Appendix Tables A4 and A5).These differences in primary dietary items between EFNP and RUNR may be due to differences in the availability (frequency or biomass) of each taxon at each site.Previous studies at Epping Forest National Park have shown that buffel grass is the dominant grass species followed by Aristida spp., Enneapogon spp., Eragrostris lacunaria, Chrysopogon fallax and the sedge Fimbristylis dichotoma.There are temporal fluctuations in FOO, and long-term drought has affected FOO of some species (Woolnough, 1998).Buffel grass increased in FOO from 8% to 48% from 1987 to 1997 and in biomass from 13% to 54% from 1987 to 1994 (Back, 1997;Horsup, 1998;Woolnough, 1998).RUNR is dominated by buffel grass and Aristida spp.
Significant seasonal differences were found in the diet of the NHW at both EFNP and RUNR.Only one study has previously investigated temporal changes in the diet of the NHW.Woolnough (1998) assessed the diet of the NHW at EFNP and found the diet remained relatively consistent throughout the year, with no clear changes in composition between seasons.However, Woolnough (1998) recognised that the methods adopted to quantify the diet were likely not adequate for detecting subtle seasonal changes.
At EFNP, all seasonal pairwise comparisons were significant ex-  and Science, 2018;Jørgensen et al., 2021), but no information regarding season or seasonal changes was provided.An assessment of pasture productivity at EFNP approximately three decades ago found productivity was low during 9 months of the year, over spring, winter and autumn, followed by a drastic increase of up to 20-fold during summer (Johnson, 1991).Determining the influence of forage availability on the diet would require the forage community to be assessed alongside an assessment of the diet.
The results of this study revealed that buffel grass dominated the diet of the NHW, and as Woolnough (1998) suggested is likely a reflection of availability of the species within the habitat.Forage availability has also been found to influence diet choice in other herbivorous species (Hopcraft et al., 2010;McNaughton & Banyikwa, 1995); for example, elephants (Loxodonta africana) increase the amount of grasses consumed seasonally as grass biomass increases within the habitat (Shannon et al., 2013).The forage communities at EFNP and RUNR were not assessed in this study, but if forage availability is determining the diet of the NHW, the dominance of buffel grass in the diet would suggest that buffel grass is readily available throughout each site.An abundance of buffel grass in the habitat is a concerning possibility, as the invasive species has been identified as a threat to native vegetation communities within NHW habitat (Department of Environment and Science, 2022), and its impact on the nutrition of the species is currently unknown.
The diet of the NHW at EFNP had not significantly differed between samples collected in past years (2013-2017) and samples collected for the current study during 2020-2021; however, this result is unreliable as only a single sample represented each past year (2013, 2014 & 2017).Although the diet in past years was not significantly different, changes in the mean abundance of some dietary taxa, including buffel grass, may indicate narrowing dietary niche breadth of the NHW at EFNP.For example, the mean abundance of buffel grass increased more than 30% from 2013-2017 to 2020-2021, while the mean abundance of three taxa decreased by approximately 10% (Table 2).It should be noted only three scat samples were available for analysis from 2013 to 2017; hence, a larger sample size would have provided a better indication of changes and trends in the diet.
The diet of the NHW at EFNP has changed considerably to that reported in past assessments.Flosser (1986) assessed the species composition (%) of the diet and listed five taxa as primary dietary items: Eragrostris lacunaria, Chrysopogon fallax, Heteropogon contortus, Fimbristylis spp.and Aristida spp.Crossman (1988) assessed species frequency within the diet (%) and identified Heteropogon contortus, Aristida spp., Enneapogon spp.and Sporobolus caroli as primary dietary items.Woolnough (1998) was the first to report buffel grass as a primary dietary item, alongside Aristida spp.and Enneapogon spp.(Table 4).The contribution of buffel grass to the diet of the NHW at EFNP has been increasing over recent decades.
were the most dominant taxa within the diet during 1998, contributing ~21% and ~17%, respectively (Woolnough, 1998), yet contributed <1% each during 2020-2021 (Table 4).Hence, our study has shown there have been temporal changes to the diet of NHW since 1986.
When comparing the diet of the NHW over time, it is important to note that different dietary assessment techniques were used.Previously, histological techniques were used to assess the diet of the NHW (Crossman, 1988;Flosser, 1986;Woolnough, 1998) whereas the current study used DNA metabarcoding.The metabarcoding approach is more comprehensive than a histological approach, with improved capability for accurately detecting dietary items that are rare or consumed in very small quantities (Nichols et al., 2016;Soininen et al., 2009;Stapleton et al., 2022).For example, our metabarcoding technique identified more taxa in the diet compared with past assessments.Some taxa detected within the diet in this study, but not previously, may have been present in the diet at the time of previous assessments but in amounts not detectable by histological analysis.Additional taxa may also be identified in the future due to the expectation that increased sequence data for more plant species will increase in available databases over time.Although bias therefore exists in the comparison of past and current results, the variations in primary dietary items over time are pronounced and thus likely do reflect true changes in the diet.Woolnough and Johnson (2000) found eastern grey kangaroos  Crossman (1988), Flosser (1986), and Woolnough (1998) and the current study.The genera included are those that contributed ≥1% to diet abundance in either Flosser (1986) and Woolnough (1998) or the current study, and every genera reported by Crossman (1988).however, the method has been recognised as capable of providing estimates (Deagle et al., 2019;Stapleton et al., 2022;Verkuil et al., 2022).Given buffel grass was detected in every sample and returned a mean RRA much greater than any other taxa, it is likely accurate that buffel grass does dominate the diet of the NHW at both sites.
Another limitation of this study is that the assessment of the diet of the NHW at EFNP is incomplete, as scat samples were not collected during autumn.Scats from all four seasons must be analysed to achieve representation of the whole diet, especially con- Scat collection occurred across nine sampling periods.Scats were collected from EFNP during four seasons: winter and spring 2020, summer 2020/2021 and winter 2021; and from RUNR during five seasons: winter 2020, summer 2020/2021 and autumn, winter and spring 2021.Between eight and 11 scat samples were obtained during each sampling period (Appendix Table Scat samples were sent to the Australian Genome Research Facility in Adelaide, Australia.DNA was extracted using the DNeasy PowerSoil Pro DNA Extraction Kit (Qiagen) according to the manufacturer's instructions.Of 85 scat samples, 77 yielded DNA of adequate quality for analysis.The presence of plant DNA from faecal samples was detected using the primer pair ITS2F (TGTGA ATT GCA RRA TYCMG) and ITS2R (CCCGH YTG AYY TGR GGTCDC; Moorhouse-Gann et al., 2018) producing a 250 bp fragment.Image analysis was performed in real time by MiSeq Control Software version 3.1.0.13 and Real-Time Analysis version 1.18.54.4.Sequencing was performed using an Illumina MiSeq platform extract DNA from eight scats, including two collected from RUNR during winter 2020, two collected during winter 2020 and three collected during winter 2021 from EFNP, and the single scat collected from EFNP during 2015.Sequencing yielded a total of 9,984,833 reads from a total of 77 scat samples (Mean = 129,673 ± 34,717 S.D.).Sequencing yielded 4,709,816 reads from 39 scat samples from EFNP (Mean = 130,828 ± 33,692 S.D.) and 4,870,462 reads from 38 scat samples from RUNR (Mean = 128,170 ± 36,906 S.D.) during 2020-2021.Three of the four DNA samples extracted from scats collected from EFNP during 2013-2017 resulted in 139,659 sequences from 2013, 157,191 sequences from 2014 and 107,705 sequences from 2017.In total, 191 taxonomic categories were identified in the diet.
Chrysopogon latifolius was the second most abundant taxon within F I G U R E 1 Frequency of occurrence (FOO) of taxa in the diet of northern hairy-nosed wombat at Epping Forest National Park during 2020-2021.Only taxa with a FOO of ≥30% are represented.the diet during winter 2020 (3.7%), followed by unidentified Fimbristylis spp.(1.0%).Chrysopogon latifolius returned the second highest abundance during spring 2020 (17.5%) and summer 2020/2021 (16.5%), followed by Boerhavia erecta (spring 2020 = 2.0%; summer 2020/2021 = 13.9%).During winter 2021, unidentified Malvaceae spp.returned the second highest abundance within the diet (1.2%), followed by Evolvulus alsinoides (1.0%; Figure 4).At EFNP, two taxa had a FOO of 100% in every season: buffel grass and unassigned spp.Buffel grass, Chrysopogon latifolius, Fimbristylis spp.and unassigned spp.were the only taxa with a FOO of F I G U R E 2 Frequency of occurrence (FOO) of taxa in the diet of northern hairy-nosed wombat at Richard Underwood Nature Refuge during 2020-2021.Only taxa with a FOO of ≥30% are represented.F I G U R E 3 Proportional abundance of taxa in the diet of northern hairy-nosed wombat at Epping Forest National Park (EFNP) and Richard Underwood Nature Refuge (RUNR) during 2020-2021.All taxa that did not contribute ≥0.5% within at least one season are combined and represented as 'Other'.100% during winter 2020 and spring 2020.Despite returning the third highest abundance, the FOO of Boerhavia erecta during spring 2020 was 33%.During summer 2020/2021, 11 taxa had a FOO of 100%, including Boerhavia erecta, Chrysopogon latifolius and Fimbristylis spp.(Appendix Table cept between the two winter seasons (2020 and 2021) and between spring 2020 and each winter season (2020 and 2021).Like EFNP, every seasonal pairwise comparison was significant at RUNR except summer 2020/2021 and each winter season (2020 and 2021).Changes in forage availability within each site could be responsible for seasonal changes in the diet.The vegetation community at RUNR has been briefly described (Department of Environment

(
Macropus giganteus) and NHW shared 85% of diet species.Sobek and Walker (2021) used DNA metabarcoding to compare the diet of the southern hairy-nosed wombat and western grey kangaroo (Macropus fuliginosus) and found 20 genera and 10 genera, respectively, with eight genera in common.Both diets included introduced and native genera.In contrast to the southern hairy-nosed wombat(Sobek & Walker, 2021), NHW consumed species in the genera Austrostipa (native), Chenopodium (native), Euphorbia (native), Mediocargo (introduced) and Oxalis (introduced).The highest occurrence of plants in southern hairy-nosed wombat scats was from the Atriplex, Carrichtera, Medicargo and Sclerolaena genera(Sobek & Walker, 2021).In the NHW diet, the introduced Oxalis corniculata was the only species identified that was also in the diet of the southern hairy-nosed wombat.Differences in wombat species distribution and floristic composition within the habitats likely account for differences in the diets of the two wombat species.A limitation of this study was the use of RRA to quantify the abundance of each taxon within the diet.Using RRA to quantifying dietary abundance has been criticised for lacking accuracy, as several biological and technical factors can affect the number of sequence reads produced for each food item(Pompanon et al., 2012); TA B L E 4 Abundance and frequency of occurrence (FOO) of each genus in the diet of the NHW at EFNP across four studies: sidering season was found to significantly affect the diet at EFNP.Aside from assessing the diet of the NHW at EFNP during all four seasons, the dietary assessment would be greatly complimented by assessment of the composition, frequency and biomass of forage available within both habitats.Such information about forage availability would assist in determining whether and how it influences diet choice in NHW.A greater understanding of what factors influence the diet of the NHW is essential for effective management of the diet, for example increasing native grasses and reducing invasive grasses.In addition, assessment of the forage community would provide insight into the spread of buffel grass throughout EFNP and RUNR, necessary information for managing and protecting the vegetation communities within NHW habitat.Our study has further confirmed the NHW is a generalist herbivore and increased the number of known preferred dietary items.This assessment of the diet of the NHW is the first since the establishment of the translocated population at RUNR.The diet of the NHW differs significantly between the populations at EFNP and RUNR and varies significantly between seasons at each site.In comparison with the diet of the NHW reported in past studies, the contributions of specific plant taxa have changed over time, including buffel grass, which has dramatically increased in frequency and abundance in the diet.Overall, buffel grass dominated the diet, with a total abundance almost 10 times greater than any other taxon.Our study has provided an updated assessment of the diet of the critically endangered NHW at EFNP and a new assessment at RUNR that will assist with species conservation.Data curation (lead); formal analysis (lead); writing -original draft (lead).Julie M. Old: Conceptualization (equal); funding acquisition (equal); methodology (equal); project administration (equal); supervision (equal); writing -review and editing (equal).Hayley Jade Stannard: Conceptualization (equal); funding acquisition (equal); methodology (equal); project administration (equal); supervision (equal); writing -review and editing Location of scat samples collected at Epping Forest National Park (EFNP) and Richard Underwood Nature Refuge (RUNR).
Number of scat samples from Epping Forest National Park (EFNP) in which each taxon was present (frequency of occurrence) during each sampling period from 2020 to 2021.Number of scat samples from Richard Underwood Nature Refuge (RUNR) in which each taxon was present (frequency of occurrence) during each sampling period from 2020 to 2021.Abundance (or RRA) of taxa in the diet of the northern hairy-nosed wombat (NHNW) at Epping Forest National Park (EFNP) throughout each sampling period from 2013 to 2021.
TA B L E 2

5
Proportional abundance of taxa in the diet of northern hairy-nosed wombat at Richard Underwood Nature Refuge, in total and across each season.All taxa that did not contribute ≥0.5% within at least one season are combined and represented as 'Other'.Pairwise SIMPER results including the 10 taxa with the greatest contribution to dissimilarity for significant pairwise ANOSIM tests from Richard Underwood Nature Refuge (RUNR).
TA B L E 3